The main long-term goal of this research is to completely understand both the requirements for RNA splicing and the role of splicing in the expression of the intron-containing T4 gene (td) coding for thymidylate synthase (TSase). The requirements will certainly include structural features of the td gene itself and will probably also involve the action of products of other genes. We will define these requirements by finding and characterizing mutations that affect RNA splicing and gene expression. A secondary goal is to understand the structure and function of the T4 TSase and the closely related enzyme dihydrofolate reductase and how their synthesis is regulated. These two enzymes have several common roles in T4-infected E. coli and their structural genes overlap. The specific aims of the proposed research are to genetically map many available mutations in the T4 td gene; to characterize td mutations, particularly those in the intron, as to their change in DNA sequence and effect on RNA splicing and gene expression; to isolate mutants with new mutations affecting RNA splicing at false revertants of td mutants defective in splicing and to characterize these new mutations; and to study the structure, function and expression of the TSase and dihydrofolate reductase gene by correlating specific base changes with specific alterations. These studies will provide new types of T4 mutants affecting RNA splicing. Characterization of these mutants will lead to a better understanding of the mechanism and the role of RNA splicing in T4-infected E. coli. A knowledge of the mechanisms involved in RNA splicing in phage-infected bacteria should be helpful in the analysis of RNA splicing in eukaryotes. It is likely that in some cases very similar mechanisms will be found.